The present invention relates generally to the fields of molecular biology and genetic engineering. More specifically, the invention provides inducible packaging cell lines for production of lentivirus vectors.
Various publications or patents may be referenced in this application to describe the state of the art to which the invention pertains. Each of these publications or patents, is incorporated by reference herein.
Retroviral vectors and packaging cells are important tools for gene transfer into eucaryotic cells. Replication-defective retroviral vectors contain the cis-acting sequences required for efficient virus replication. The packaging cell lines provide viral proteins required in trans for virus replication. Introduction of a retroviral vector into a suitable packaging cell enables the propagation of vector virus in the absence of replication-competent virus.
Most retroviral vectors have been derived from oncoviruses, such as murine leukemia virus (MLV). A limitation of oncovirus-based systems is that proviral integration into the host genome requires at least one round of cell division.
In contrast, the preintegration complex of the lentivirus, human immunodeficiency virus-1 (HIV-1), can integrate into the genomes of non-dividing cells. This feature, among others, of HIV-1 has provided incentive for the development of HIV-1 packaging cell systems for use in in vivo gene therapy. In addition, HIV-1 packaging cell lines can be used to study HIV-1 replication, mutation frequencies and latency.
The HIV-1 genome codes for the proteins Gag, Pol, and Env which are common to all retroviruses. In addition, it contains two regulatory genes, tat and rev, which are important for efficient viral replication, and four accessory genes, vif, vpr, spit, and nef, which have been demonstrated to, be dispensable for HIV-1 vector production from transfected cells.
Several biological features of HIV-1 are obstacles to the development of safe, efficient HIV-1 based packaging lines. For instance, the pathogenicity associated with the virus requires that viral stocks be completely void of replication-competent virus. Also, as mentioned, HIV-1 expresses several accessory proteins reported to enhance high-level infection and replication. In some instances (e.g., for drug screening systems), it would be desirable to include them in packaging cell lines. However, several of the trans-acting proteins, including Env, have been reported to be cytotoxic or cytostatic, hampering the establishment of a constitutively expressing packaging cell line.
One way to circumvent the cytotoxicity of constitutively-expressed HIV-1 accessory proteins is to develop a system for inducibly expressing the proteins. Furthermore, inducible packaging cells provide a safe system for drug screening, since viral proteins are not produced until induction, and biologically hazardous wild-type virus is never generated.
Inducible HIV-1 packaging cell lines have been developed. For instance, Yu et al. (J. Virol. 70: 4530-4537, 1996) reported an HIV-1 packaging cell line that utilized the tetracycline-inducible expression system to control the expression of the Rev regulatory protein, which in turn controls expression of late proteins, including Gag, Pol and Env. Kaul et al. (Virology 249: 167-174, 1998) reported a tetracycline-inducible HIV-1 packaging cell line which, unlike its predecessor (Ku et al, 1996, supra), was capable of expressing all HIV-1 structural, regulatory and enzymatic proteins, but lacked a majority of the cis-acting sequences. Kafri et al. (J. Virol. 73: 576-584, 1999) reported the development of a VSV-G pseudotyped, tetracycline-inducible HIV-1 packaging cell line, which further expressed the Green Fluorescent Protein (GFP). The use of VSV-G bolsters virus titers, but concomitantly results in loss of target cell specificity.
The tetracycline-inducible system is effective, but has limitations. For instance, establishing appropriate expression levels for the DNA constructs used in the system can be difficult, due to the toxicity associated with the transactivation protein utilized in the induction system. This protein must be expressed in sufficient quantity to effect adequate induction of the HIV-1 proteins, but production of too much of the protein is toxic to the cells.
Thus, improvements in lentivirus packaging cell lines are needed and would advance the art of lentivirus based gene therapy and assay systems. Such improvements include inducible systems that are easy to establish and use, and that produce higher vector titers that those obtainable with current systems.
According to one aspect of the present invention, a transgenic mammalian packaging cell line for producing lentiviral vectors is provided. The cell line comprises (a) a DNA construct expressing a transcription factor responsive to ecdysone or an analog of ecdysone; and (b) one or more DNA constructs comprising one or more selected lentivirus genes required for packaging of a lentiviral vector; and a gene encoding a viral envelope protein. The lentivirus genes and the gene encoding the viral envelope protein are operably linked to at least one DNA response element acted upon by the transcription factor, and the cell line is transfectable with the lentiviral vector. The cell line preferably produces HIV-1 based vectors, and preferably is generated from 293T cells.
In one embodiment, the cell line includes a gene encoding an HIV-1 envelope protein. In another embodiment, the cell line includes a gene encoding a VSV-G envelope protein. In certain embodiments of the HIV-1 based cell line, the line expresses HIV-1 core genes, accessory genes and regulatory genes. In particularly preferred embodiments, the HIV-1 core and accessory genes and the HIV-1 env gene are introduced into the cell line on separate vectors. In other embodiments, the cell line is deleted for at least one HIV-1 accessory gene or regulatory genes, and preferably is deleted for all HIV-1 accessory genes except rev.
According to another aspect of the invention, a method for producing a lentivirus vector stock is provided. The method utilizes the above-described packaging cell line and comprises transfecting the cell line with the lentivirus vector and exposing the transfected cell line to ecdysone or an analog of ecdysone (preferably ponasterone A), for a time and under conditions enabling activation of the transcription factor and resultant induction of expression of the genes operably linked to the DNA response element. Thus, the proteins required for packaging of the lentiviral vector into virus particles are generated, resulting in production of the lentivirus vector stock.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed descriptions and examples that follow.